Thrombin plays a central role in the coagulation cascade of higher animals. The primary function of thrombin is to activate fibrinogen to fibrin and generate an insoluble fibrin clot. It also serves regulatory functions in coagulopathy by activating several participating cofactors and proteases such as factor V, factor VIII, factor XIII and protein C. In a pathologic state, thrombin promotes coagulopathy, activates platelets and causes secretion of granular substances that exacerbate the condition. Thrombin's interaction with endothelial cells, smooth muscle cells, fibroblasts, and monocytes/macrophages contribute further to the inflammatory process in thrombotic events. An acute blockage of a coronary artery by a thrombus causes a myocardial infarction. In its early stages, the condition may be alleviated with thrombolytic therapy. However, typical thrombolysis with tissue plasminogen activator, urokinase or streptokinase is problematic. Acute thrombotic reocclusion often occurs after initial successful thrombolysis using these agents. Although the mechanism of reocclusion has not been clearly elucidated, thrombus-bound thrombin may contribute to this problem. Potent and specific agents that neutralize thrombus-bound thrombin would be desirable.
Thrombin is a member of the trypsin family of serine proteases. In addition to the catalytic triad (Asp 102, His 57 and Ser 195) a feature common to the active site of all serine proteases, asp 189 in the primary substrate binding site (S1) of the trypsin family plays an important role in the recognition and binding of substrates and inhibitors.
A natural anticoagulant, heparin inhibits thrombin through a mechanism requiring a heparin-antithrombin III compounds. Heparin is known to be poorly accessible to thrombus-bound thrombin. Furthermore, heparin often causes bleeding when used therapeutically and is unable to prevent the occlusive complications in atherosclerotic vascular diseases or reocclusion following successful thrombolysis.
Another agent known to be effective for the inhibition of thrombus-bound thrombin is hirudin. Hirudin is produced by the salivary glands of the European medicinal leech Hirudo medicinalis and is a small protein of 65 amino acid residues. It has several potential advantages over other antithrombotics. It is the most potent and specific thrombin inhibitor known having a K.sub.i value of 2.2.times.10.sup.-14 M. Hirudin blocks the active site (AS) and the fibrinogen recognition exosite (FRE) of thrombin simultaneously. Hirudin also inhibits thrombus-bound thrombin as well as circulating thrombin and it has a long half-life of 30-60 minutes when given intravenously or subcutaneously, depending on the species. Hirudin has very weak antigenicity, and it has no reported acute side effects following intravenous or subcutaneous administration.
Synthetic thrombin inhibitors based on the hirudin sequence offer an advantage over native hirudin. They mimic the distinctive mechanism of hirudin and are more readily available through chemical synthesis. The crystal structure of the human a-thrombin/hirudin complex reveals that hirudin interacts with the enzyme through an active site inhibitor domain (hirudin.sup.1-48), a FRE inhibitor segment (hirudin.sup.55-65), and a linker segment (hirudin.sup.49-54) which connects these binding components.
The bulky active site inhibitor segment, hirudin .sup.1-48, is sufficiently large and serves to obstruct the enzyme surface. This action has been shown to be simulated when hirudin .sup.1-48 is replaced by a small active site inhibitor segment, D-Phe-Pro-Arg-Pro, with some loss in inhibitory potency (Maraganore, J. M., Bourdon, P., Jablonsky, J., Ramachandran, K. L., & Fenton, J. W. 11 (1990) Biochemistry 29, 7095-7101; DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. Ni, F., and Konishi, Y., (1990) J.Biol.Chem 265, 21698-21703; Bourdon, P., Jablonski, J. -A., Chao, B. H., and Maraganore, J. M., 9, (1991) (FEBS Lett. 294, 163-166).
Investigators have focused on the use of D-Phe-Pro-Arg-Pro or its analog in the design of active site inhibitors. The crystal structure of D-Phe-Pro-Arg chloromethylketone (PPACK)-thrombin suggested that the D-Phe-Pro-Arg-Pro in bivalent inhibitors bind to the thrombin active site in a substrate binding mode, wherein Arg-X is the scissile peptide bond. The active site inhibitor segment, D-Phe-Pro-Arg-Pro, of the bivalent inhibitors is known to be hydrolyzed slowly by thrombin (DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J., Ni, F. and Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703; Witting, J. I., Bourdon, P., Maraganore, J. M., and Fenton, J. W., II (1992) BioChem. J. 287, 663-664). The amino acids (D-Phe)-Pro-Arg comprised in the substrate type inhibitor (D-Phe)-Pro-Arg-Pro respectively bind to the S3, S2 and S1 subsites of thrombin.
Hirulog-8.TM. is a bivalent thrombin inhibitor composed of the substrate type inhibitor (D-Phe)-Pro-Arg-Pro, and the native sequence of the hirudin exosite segment 52-65 both linked through a suitable linker (Maraganore et al. U.S. Pat. No. 5,196,404). Since the structure of those thrombin inhibitor is very similar to the structure of hirudin, the interactions of the substrate type active site inhibitor with thrombin are the same as the interactions between the active site of hirudin and thrombin. In addition, it has been shown that the portion (D-Phe)-Pro-Arg-CO can be used in a bivalent thrombin inhibitor (DiMaio et al. International publication WO 91/19734). Apparently, the use of the acetyl function at the scissile position gives more resistance to enzyme degradation without affecting the inhibitory activity. The scissile position in a substrate is a position that is recognised by the enzyme and where the hydrolysis takes place. It is therefore advantageous to eliminate or to modify the scissile position in order to give to more resistance to enzyme degradation. Since the structure of the two classes of bivalent thrombin inhibitors mentioned above are similar to the structure of hirudin, their synthesis is difficult, cumbersome, uses dangerous chemicals and affords low yields of the desired compounds. There is therefore a need for other thrombin inhibitors that would combine high inhibiting activity, enzyme resistance and affordable synthesis.
Besides substrate-type inhibitors, nonsubstrate type inhibitors could be designed to block the active site of thrombin without being cleaved. Examples of these may be derived from arginine and benzamidine to give, for example, (2R,4R)-4-methyl-1-[N.sup..alpha. -(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulphonyl)-L-arginyl]-2-piperidin e carboxylic acid (MD-805), N.sup..alpha. -(4-toluene-sulphonyl)-D,L-amidinophenylalanyl-piperidine (TAPAP), and N.sup..alpha. -(2-naphthyl-sulphonyl-glycyl)-D-L,p-amidinophenylalanyl-piperidine (NAPAP). These active-site directed synthetic inhibitors have a short half-life of less than several minutes in the circulation. This activity is not of sufficient duration to be effective against the continuous production of thrombin by the patient or against the effect of liberated thrombus bound-thrombin. The characteristic sequence of these compounds starting from the N-terminus is an aromatic group, arginyl or benzamidyl, and piperidide or its analogs. In contrast to hirudin-based sequences, these moieties would be expected to occupy the S3, S1 and S2 subsites of the thrombin active site, respectively.
This mechanism of interaction contrasts with the mode of interaction manifest by substrate-like inhibitors. Accordingly, incorporation of a non-substrate type active site inhibitor into the bivalent inhibitor may have advantages over the substrate like counterparts. For example, a linker attached to the P2 residue piperidide or its analogs could eliminate a labile peptide bond that normally spans the scissile position. The potency of the bivalent inhibitor might be improved because of the higher affinity of the non-substrate type active site-directed segment.
It would be desirable to develop a shortened thrombin inhibitor of the hirudin type. Such a shortened sequence would be easier to synthesize and cheaper to produce. It would have a drastically shortened linear sequence and would be less subject to enzymatic degradation in a mammal.
It has been found that such a hirudin-like agent would more likely work well if it blocked both the enzyme activity site of thrombin and the fibrinogen-recognition exosite. It would be even more desirable if both these sequences were chemically connected so as to have both abilities in one compound.
It has been previously reported that the combination of dansyl or dansyl analogues, arginine or benzamidine, and pipecolic acid attaches to the thrombin enzyme activity site. But it has been shown that such activity is weak and not pharmacologically useful (James C. Powers and Chih-Min Kam, Thrombin: Structure and Function, Chapter 4, (1992), Lawrence J.Berliner. Plenum Press, New York).
The invention seeks to provide improved bivalent inhibitors having increased potency and proteolytic stability comprising non-substrate type active site inhibitor segment.
Abbreviations. The following abbreviations have been used in the specification: Abu, .gamma.-aminobutyric acid; Ac, acetyl; Aca, .epsilon.-aminocaproic acid; Aca*, 8-aminocapylic acid; Acha, 1-aminocyclohexane-carboxylic acid; Ada, 12-aminododecanoic acid; AMC, 7-amino-4-methylcoumarin; Aua, 11-aminoundecanoic acid; Ava, .delta.-aminovaleric acid; Bal, .beta.-alanine; Boc, tert-butyloxycarbonyl; BrBzs, 4-bromobenzenesulfonyl; Bzs, benzene sulfonyl; Cha, .beta.-cyclohexylalanine; Fmoc, 9-fluorenylmethoxycarbonyl; FRE, fibrinogen recognition exo site; HPLC, high performance liquid chromatography; MD805, (2R,4R)-4-methyl-1-[N.sup..alpha. -(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulphonyl-glycyl)-L-arginyl]-2-pi peridine carboxylic acid; NAPAP, N.sup..alpha. (2-naphtyl-sulphonyl)-D,L-p-amidinophenylalanyl-piperidide; Nas, naphtylsulfonyl; Nle, norleucine; 3-TAPAP, N.sup..alpha. -(4-toluene-sulphonyl)-D,L-p-amidinophenylalanyl-piperidide; OBzl, benzylester; Pip, pipecolic acid; PPACK, D-Phe-Pro-Arg chloromethylketone; tBbs, 4-tert-butylbenzenesulfonyl; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; TipBs, 2,4,6 triisopropylbenzenesulfonyl; TFA, trifluoroacetic acid; Tos, tosyl; Tris, 2-amino-2-(hydroxymethyl)-1,3-propanediol. All amino acid residues are L-configuration unless otherwise indicated. IC.sub.50 is defined as the inhibitor concentration required to double the clotting time relative to the control; means of three determinants .+-.SEM.